ABSTRACT The sequencing of the human genome led to a rapid cataloguing of genes and expressed sequence tags of higher organisms, including humans. However, the challenge still remains to understand the function of gene products in different biological contexts including the differentiation of adult or embryonic stem cells. This proposal seeks to address this challenge in the context of stem cell senescence and differentiation. In Aim 1 we will develop a novel, high throughput strategy to discover regulators of stem cell differentiation using lentiviral reporters, genome-wide lentiviral shRNA libraries and next generation sequencing. We will use this strategy to discover which genes and pathways may mediate differentiation of senescent or rejuvenated stem cells in which the differentiation potential has been restored. The resulting hits will be individually tested in Aim 2 by employing the LentiViral Array (LVA) platform that we developed in our laboratory to monitor stem cell differentiation with concomitant gene knockdown. To this end, we will use a novel lentiviral vector that was developed in our laboratory and enables regulatable gene knockdown with simultaneous monitoring of stem cell differentiation. In Aim 3 we will develop novel microtissue arrays to test the shRNA hits on the function of 3D microtissues prepared with senescent or rejuvenated stem cells. The proposed work is expected to generate a technological framework to facilitate understanding of the regulatory networks at work during stem cell senescence and its effects on differentiation. It may also discover novel gene targets that may reverse the effects of cellular senescence, thereby enhancing the potential of stem cells for regenerative therapies.